Spectrophotometry refers to the measurement of the absorption or transmission of incident light through solutions of test compounds. Typically, compounds of interest have characteristic spectra, transmitting or absorbing specific wavelengths of light, which can be used to determine the presence of these compounds or measure their concentration in test samples. Instruments designed for spectrophotometric absorption have a light source, for which the emitted wavelength is known and may be adjusted, and one or more detectors sensitive to desired wavelengths of transmitted or reflected light. Spectrophotometric absorption can be used to determine the amount of a given compound that is present in a test sample.
Circular dichroism (CD) is a special type of absorption method in which the molecular composition of the analyte results in differential absorption of incident light not only at a specific wavelength but also of a particular polarization state. Circular dichroism is a chiroptical method which allows one to differentiate between different enantiomers, that is, optical isomers having one or more asymmetric carbon atom (chiral) centers. When utilizing CD, generally a sample is illuminated by two circularly polarized beams of light traveling in unison. Both beams pass through the sample simultaneously and are absorbed. If the sample is optically active, the beams are absorbed to different extents. The differences in absorption of the beams can then be displayed as a function of the wavelength of the incident light beam as a CD spectrum. No difference in absorption is observed for optically inactive absorbers so that these compounds are not detected by a CD detecting system. The use of CD as a chiroptical method has been fully described in scientific literature (1).
Early applications of the CD method primarily dealt with elucidation of molecular structures, especially natural products for which a technique capable of confirming or establishing absolute stereochemistry was critical. However, CD has also reportedly been used in a clinical method to quantitatively determine unconjugated bilirubin in blood plasma (2). In the method disclosed, a complex was formed between bilirubin and human serum albumin as a CD probe for bilirubin analysis.
Clinical applications of circular dichroism are also discussed by Neil Purdie and Kathy A. Swallows in Analytical Chemistry, Vol. 61, No. 2, pp 77A-89A (1989), herein incorporated by reference. Possible clinical applications of CD are disclosed to include measurement of cholesterol levels and detection of anabolic steroids. However, suitable chemical reagents for carrying out such testing are not disclosed.
Regarding the use of spectrophotometric absorption, fluorescence, derivative spectrophotometry or CD methods herein disclosed to measure cholesterol levels, it is noted that the population at large is continually advised that it is prudent to know serum cholesterol levels and constantly reminded that an uncontrolled diet and a lack of exercise can lead to accumulation of arterial plaque that will increase the risk of atherosclerosis and coronary heart disease. Statistical studies have shown that other risk factors, such as age, gender, heredity, tobacco and alcohol consumption, etc. must also be considered when counselling patients about the risks (3,4).
The magnitude of the program for screening the general public is so immense that automated methods for cholesterol determinations are necessary. The tests currently used differ in complexity from the simple dip-stick approach, which uses a color sensitive reaction on a paper support, to the sophisticated lipid profile tests, in which the distribution of cholesterol among the various solubilizing molecular species is determined (5). The dip-stick is only a preliminary qualitative test upon which a decision for the fuller, more quantitative measurement can be based.
At the conclusion of a recent extensive study of how health risk factors are related to elevated levels of serum cholesterol, a report (6) was prepared by the Laboratory Standardization Panel (LSP) of the National Cholesterol Education Program (NCEP) in which the measure of risk was correlated with three ranges of total cholesterol (TC): low risk if less than 200 mg/dL; moderate risk in the range 200-239 mg/dL; and high risk if greater than 240 mg/dL. In order to place a particular individual into one or other of these categories, all that is required is a serum TC measurement. The other risk factors (3,4) are then added as a basis for further patient counselling. This relatively simple approach replaces an earlier recommendation (3,7), in which relative risk was established using a ratio of TC to high density lipoprotein cholesterol (HDL-C) equal to 5. A ratio lower than 5 implies a high level of HDL-C and a low relative risk. For this diagnosis, HDL-C is measured in a second independent test.
The same report (6) hastened to add that there were serious inaccuracies in measurements made by numerous clinical laboratories in the determination of the amount of TC present in human serum reference standards.
Statistically the results showed that, in data from 1500 laboratories, 47% failed to measure the true value to within a coefficient of variance (CV) of .+-.5%, and 18% of these failed at a CV of .+-.10%. As a consequence, the LSP recommended that an improvement in CV to within .+-.3% for TC should be achieved by 1992. Recent surveys indicate that certified laboratories are well on their way to meeting that challenge, using the current clinical methods and instrumentation (8). The LSP did not report the inaccuracies associated with the determination of the distribution of cholesterol among the various lipids and lipoproteins, but did indicate that an evaluation would be made in the future. The very poor proficiency and lack of reliability in the measurement of serum or plasma HDL-C, has been eloquently described in three recent publications (7,9,10), where interlaboratory CV's as high as 38% were reported (9). A 1987 evaluation by the College of American Pathologists (CAP) of the measurement of the same sample for HDL-C by over two thousand laboratories showed, that more than one third differed by more than 5% from the reference value. Interlaboratory CV's among groups using the same method did improve to 16.5%, but it is still too imprecise to be of any predictive clinical value. This is the reason the TC:HDL-C ratio is no longer used in risk assessment, although it offers potential advantages in defining the true clinical picture.
Regarding the presently used lipid profile studies, cholesterol is distributed in the serum mainly associated with high density lipoprotein (HDL-C) and low density lipoprotein (LDL-C) fractions and with triglycerides as the very low density lipoprotein cholesterol (VLDL-C) fraction. There is plenty of statistical evidence from a number of long term clinical tests to justify that a high proportion of HDL-C and a low proportion of LDL-C is associated with lower relative risk (3,4) or in simpler terms, high levels of LDL-C are to be avoided where possible. HDL-C is beneficial, provided the level is not excessively low, i.e., less than 30 mg/dL (7). VLDL-C cholesterol has not been implicated in any risk determination, but high triglyceride itself can be a serious problem. In a typical lipid profile study, total cholesterols are measured directly and HDL-C is measured in the supernatant remaining after treatment of the sample with an agent to precipitate out LDL-C and VLDL-C. VLDL-C is taken to be a fixed fraction (e.g, 0.2) of the triglyceride, which is also measured directly in a separate assay. LDL-C is calculated from these figures and is not measured directly. The propagation of errors in each of the three independent measurements makes LDL-C the fraction known with least overall accuracy and precision, although it may be the most significant aspect of cardiovascular risk. Because of this, it is difficult to meaningfully monitor and justify that clinical progress has been made in LDL-C reduction therapy with time.
At a workshop and subsequent roundtable session (13,14) held at the 43rd Meeting of the American Association for Clinical Chemistry, the present state of the art in this area was summarized. It was concluded that accuracy is essential in HDL measurement. While presently available precipitation methods can give satisfactory results, the values obtained by these methods in routine clinical laboratory settings do not meet the real medical needs. The CAP comprehensive chemistry proficiency survey from 1982 to 1991 for HDL-C showed interlaboratory CVs of about 20% in 1991, with no overall improvement since 1982. The CVs delivered by clinical instruments used for HDL-C measurements ranged from 7.6% for the Dimension to 50% for the Ektachem. At the sessions, it was also noted that direct methods for LDL cholesterol are needed. The use of triglyceride determinations to estimate VLDL-C by the Friedewald equation is the method of choice now until better methods become available. To quote the workshop syllabus, "The variability typically observed in the measurement of total and HDL cholesterol and triglycerides may preclude attaining acceptable precision. In fact, to achieve the ideal precision in LDL cholesterol estimation, the precision of the constituent measurements must be better than their ideal specifications."